Calcium release-activated Calcium (CRAC) current: Impact on human B-cell function and its physiological role in human mesothelial cells

钙（Ca 2+）是一种普遍存在的信号信使，在细胞的生命和死亡的各个方面都起着至关重要的作用。细胞可以通过从细胞内储存的Ca 2+释放或通过打开质膜（PM）中的Ca 2+传导通道来增加其细胞内Ca 2+浓度，所述Ca 2+传导通道例如由通道形成蛋白奥赖（希腊神话中的天堂之门的守护者）和内质网（ER）Ca 2+传感器基质相互作用分子（STIM）组成的钙释放激活的钙通道（CRAC）。在储存耗尽时，STIM结合并激活质膜定位的奥赖通道，提供几乎所有真核细胞中正常细胞功能所必需的Ca 2+内流途径。因此，仔细滴定STIM和奥赖的蛋白浓度确定了Ca 2+内流的量及其随后的下游效应，例如活化T细胞的核因子（NFAT）基因转录的信号传导级联、促凋亡途径如EK信号传导或存活途径如B/Akt。通道形成蛋白奥赖以三种同源物Orai 1、2和3存在。钙敏感蛋白STIM作为两个同源物STIM 1和2存在。研究表明，钙库操纵的钙离子进入（SOCE）在免疫细胞功能中起着至关重要的作用，其调节异常可导致免疫缺陷、自身免疫和癌症的发生。近年来，Orai 1的生理作用得到了广泛的研究，但Orai 2或Orai 3在原代细胞中的作用仍然是未知的。因此，我建议研究Orai 2在原代人B细胞和B细胞系中的生理作用（Daudi，Raji）。人B细胞表现出描绘天然Orai 2功能的完美模型系统，表达显著量的Orai 2并且易于分离和处理。我将表征奥赖介导的Ca 2+内流与电生理，Fura 2为基础的成像和沉默的方法，并研究其生理作用的抗体生产或白细胞介素分泌，如IL-10。该项目的这一部分将获得我们关于Orai 2的一般知识和关于人类B细胞中的CRAC/SOCE的有限知识（SA 1）。Ca 2+信号传导和CRAC电流在我的第二个研究项目中也起着至关重要的作用。TNF α和NF-κ B信号通路都是Ca 2+依赖性过程。Haining Yang等人能够确定这些信号通路在石棉诱导的间皮瘤发生中的重要性，间皮瘤是一种特殊形式的癌症，在美国每年导致约2.000-3.000人死亡。潜在的Ca 2+动员过程仍然是难以捉摸的，一般来说，它几乎是一无所知的人间皮细胞（HM）的Ca 2+信号。 因此，我建议在原代人间皮细胞的SOCE和CRAC的特点，并阐明这一特定的Ca 2+进入途径的石棉诱导的肿瘤发生（SA 2）的发展的贡献。

Calcium (Ca2+) is an ubiquitous signalling messenger and plays an essential role in every aspect of a cell s life and death. Cells can increase their intracellular Ca2+ concentration either by releasing Ca2+ from intracellular stores or by opening of Ca2+ conducting channels in the plasma membrane (PM), such as the Calcium release-activated calcium channel (CRAC) composed of the channel-forming protein Orai (after keeper of heaven's gate in Greek mythology) and the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule (STIM). Upon store depletion, STIM binds and activates plasma membrane localized Orai channels, providing a Ca2+ influx pathway that is essential for proper cell function in almost all eukaryotic cells. Careful titration of the protein concentration of STIM and Orai thus determines the amount of Ca2+ influx and its subsequent downstream effects e.g. nuclear factor of activated T cells (NFAT) signalling cascades of gene transcription, proapoptotic pathways such as EK signalling or survival pathways such as B/Akt. The channel forming protein Orai exists as the three homologs Orai1, 2 and 3. The calcium sensing protein STIM exists as the two homologs STIM1 and 2. It is already shown that store operated calcium entry (SOCE) plays a crucial role in immune cell function and that dysregulation in this process leads to the development of immunodeficiency, autoimmunity and cancer.While the physiological role of Orai1 was intensively researched within the last years, the impact of Orai2 or Orai3 function in primary cells remains still elusive. Thus, I propose to investigate the physiological role of Orai2 in primary human B-cells and B-cell lines (Daudi, Raji). Human B-cells exhibit a perfect model system for the delineation of the native Orai2 function, expressing remarkable amounts of Orai2 and easy to isolate and to handle. I will characterize the Orai mediated Ca2+ influx with electrophysiological, Fura2-based imaging and silencing approaches and investigate its physiological role regarding the antibody production or the interleukine secretion e.g. IL-10. This part of the project will gain our limited knowledge about Orai2 in general and about CRAC/SOCE in human B-cells (SA 1). Ca2+ signalling and the CRAC current play also a crucial role in my second research project. The TNFalpha and NF-kappaB signalling pathways are both Ca2+ dependent processes. Haining Yang et al. were able to identify the importance of these signalling pathways in the genesis of asbestos-induced mesothelioma, a special form of cancer, which causes ~2.000-3.000 deaths per year in the U.S.. The underlying Ca2+ mobilizing processes are still elusive and in general it is almost nothing known about Ca2+ signalling in human mesothelial cells (HM). So I suggest to characterize SOCE and CRAC in primary human mesothelial cells and to elucidate the contribution of this specific Ca2+ entry pathway to the development of asbestos-induced oncogenesis (SA 2).